Gene delivery of pDNA, antisense oligonucleotides and shRNA offers the potential for the treatment of devastating disorders including neurodegenerative diseases and spinal cord injuries, for which there are currently few treatment options. However, nucleic acid-based therapeutics is still in the early stages of development as a new category of biologics. The efficacy of gene delivery requires delivery of these molecules to the interior of the cell, presenting significant challenges for delivery strategies. Given the disadvantages associated with clinical application of viral carriers, as existing non-viral carriers transfect differentiated neurons poorly, non-viral gene delivery serves as an attractive alternative due to reduced immunogenicity, the ability to accommodate large size of transgenes, improved safety, and ease of manufacturing.
Polyethylenimine (LPEI), an off-the-shelf transfection agent, has been used to transfect a variety of cell types, including neurons in vivo and in vitro. It is thought that LPEI condenses DNA into nanoparticles, along with the cationic property of LPEI, facilitates entry of these vectors into cells by binding to negatively charged heparan sulfate proteoglycans on the cell surface. Following internalization, LPEI/DNA nanocomplexes are thought to be transported to the perinuclear region of cells. LPEI is then hypothesized to escape endosomes through a “proton-sponge effect”, releasing the DNA from the polymer and the DNA subsequently taken up into the nucleus.
Recent efforts to increase transfection efficiency and cell viability of differentiated neuron by optimizing protocols using LPEI have met with limited success. Attempts have been made to identify underlying mechanisms limiting high transfection efficiency in non-neuronal cells, primary neurons and neuronal cell-lines. Poor transfection of non-dividing, post-mitotic cells including neuronal cells is often thought to be due to the presumed inability of the pDNA in nuclear translocation. In addition, internalization and intracellular barriers were thought to restrict transfection in differentiated neuronal cell. By increasing uptake, more neuronal cells were found to express transgene, but only marginally (from 2% to 6% of total cell population). To date, the goal of attaining high transfection efficiency in differentiated neuron using non-viral carriers remains elusive and efforts to produce even more novel polymers with such properties continues.
Despite significant improvements in diagnosis and innovations in the treatment for various devastating diseases, such as cancer, autoimmune disease, and neurodegenerative disease, effective treatment of these disorders still presents major challenges. Presently, low transfection and delivery efficiencies limit the application of drug-gene therapeutics. It is this unmet need that requires the development of methods to enhance gene delivery ex vivo and in vivo and the subsequent development of galenics using this technology.
Accordingly, it is an aim of the present disclosure to ameliorate the above-mentioned disadvantages and provide an improving transfection efficiency.